Am. J. Trop. Med. Hyg., 91(3), 2014, pp. 471–480doi:10.4269/ajtmh.14-0119Copyright © 2014 by The American Society of Tropical Medicine and Hygiene

Controlled Human Malaria Infection of Tanzanians by Intradermal Injection of Aseptic,

Purified, Cryopreserved Plasmodium falciparum Sporozoites

Seif Shekalaghe, Mastidia Rutaihwa, Peter F. Billingsley, Mwajuma Chemba, Claudia A. Daubenberger, Eric R. James,Maximillian Mpina, Omar Ali Juma, Tobias Schindler, Eric Huber, Anusha Gunasekera, Anita Manoj, Beatus Simon,

Elizabeth Saverino, L. W. Preston Church, Cornelus C. Hermsen, Robert W. Sauerwein, Christopher Plowe, Meera Venkatesan,Philip Sasi, Omar Lweno, Paul Mutani, Ali Hamad, Ali Mohammed, Alwisa Urassa, Tutu Mzee, Debbie Padilla,

Adam Ruben, B. Kim Lee Sim, Marcel Tanner,† Salim Abdulla,† and Stephen L. Hoffman*†Ifakara Health Institute, Bagamoyo Research and Training Centre, Bagamoyo, Tanzania; Sanaria Inc., Rockville, Maryland; Protein Potential LLC,

Rockville, Maryland; Swiss Tropical and Public Health Institute, Basel, Switzerland; University of Basel, Switzerland; Radboud UniversityNijmegen Medical Center, Department of Medical Microbiology, Nijmegen, The Netherlands; Howard Hughes Medical Institute and Center for

Vaccine Development, University of Maryland School of Medicine, Baltimore, Maryland; Muhimbili University of Health andAllied Sciences, Dar es Salaam, Tanzania

Abstract. Controlled human malaria infection (CHMI) by mosquito bite has been used to assess anti-malaria inter-ventions in > 1,500 volunteers since development of methods for infecting mosquitoes by feeding on Plasmodiumfalciparum (Pf) gametocyte cultures. Such CHMIs have never been used in Africa. Aseptic, purified, cryopreserved Pfsporozoites, PfSPZ Challenge, were used to infect Dutch volunteers by intradermal injection. We conducted a double-blind, placebo-controlled trial to assess safety and infectivity of PfSPZ Challenge in adult male Tanzanians. Volunteerswere injected intradermally with 10,000 (N = 12) or 25,000 (N = 12) PfSPZ or normal saline (N = 6). PfSPZ Challengewas well tolerated and safe. Eleven of 12 and 10 of 11 subjects, who received 10,000 and 25,000 PfSPZ respectively,developed parasitemia. In 10,000 versus 25,000 PfSPZ groups geometric mean days from injection to Pf positivity by thickblood film was 15.4 versus 13.5 (P = 0.023). Alpha-thalassemia heterozygosity had no apparent effect on infectivity. PfSPZChallenge was safe, well tolerated, and infectious.

INTRODUCTION

Controlled human malaria infection (CHMI), intentionalinfection of subjects with malaria parasites, has been usedfor treating patients with syphilis1,2 and in research for nearlya century.3 Since the development in the 1980s of methods forinfecting mosquitoes by feeding on Plasmodium falciparum(Pf) gametocyte cultures,4–6 CHMI has been used repeatedlyand successfully in more than 1,500 volunteers in the UnitedStates and Europe.6–10 Africa suffers the most morbidity andmortality from malaria, and thus could benefit significantlyby using CHMI to facilitate development of new vaccines, drugs,and diagnostics for malaria, and for understanding innate andacquired resistance to the parasites that cause malaria. How-ever, until now such CHMIs had never been used in Africa.There are a number of reasons why CHMI has not been

established in Africa. One is that the phase 1 clinical trialfacilities and teams necessary to safely and professionallycarry out such trials have not been available until recentlyfor such studies. A second reason is that from 1985 to 2010,all CHMI studies in which volunteers were infected withPf sporozoites were conducted by exposure to the bites ofPf-infected Anopheles mosquitoes not native to Africa, andproduced in high security facilities difficult to establish,run, and maintain in Africa. To address the first limitation,we established a phase 1 clinical trial center at the IfakaraHealth Institute (IHI), Bagamoyo, Tanzania. At the sametime it became possible to manufacture aseptic, purified,cryopreserved Pf sporozoites (PfSPZ) that are highly infec-tious, a product called PfSPZ Challenge.11–13 When youngadult Dutch volunteers were injected intradermally (ID) with

doses of 2,500, 10,000, or 25,000 PfSPZ (divided intotwo 50 mL injections), five of six volunteers developedparasitemia in all three groups, and the time from injec-tion of PfSPZ Challenge to detection of parasites by thickblood smear was ~13 days in all three groups. Thus, therewas infection, but no dose response, presumably becauseincreasing the dose did not increase the numbers of spo-rozoites that exited the skin, entered the circulation, andinvaded hepatocytes.12

To begin the process of understanding how to use PfSPZChallenge in Africans, we conducted a double-blind, placebo-controlled trial to assess the safety and infectivity of ID-administered PfSPZ Challenge in 30 male, highly educated,Tanzanian residents of Dar es Salaam, Tanzania, who hadhad minimal exposure to Pf malaria during the previous5 years. As a bridge to the Dutch study, one group receivedthe regimen used in one of the Dutch groups; 5,000 PfSPZ in50 mL were injected ID into the deltoid area of both upperarms for a total of 10,000 PfSPZ.12 In a second group weincreased the dose to 25,000 PfSPZ, and based on findings inmurine model systems, which suggested ways to improve theefficiency of ID injections,14 we divided the total dosage intofour injections of 6,250 PfSPZ, each in 10 mL.As in Dutch12 and British13 subjects, PfSPZ Challenge was

safe, well tolerated, and infectious in young adult Tanzanianmales. In the bridging group the infection rate, but not thepre-patent period, was comparable to that observed for thesame dose in young adult Dutch subjects. These finding pro-vide the foundation for using CHMI with PfSPZ Challengeto assess the protective efficacy of antimalarial vaccines anddrugs in Africa.

MATERIALS AND METHODS

Study design and population. This single center, double-blind,randomized, controlled trial was conducted in Bagamoyo,

*Address correspondence to Stephen L. Hoffman, Sanaria Inc.,9800 Medical Center Drive, Rockville, MD 20850. E-mail: slhoffman@sanaria.com†These authors contributed equally to this study.

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Tanzania between February and August 2012. Thirty healthymale volunteers 20 to 35 years of age were recruited fromhigher learning institutions in Dar es Salaam. Screening foreligibility took place at the newly established BagamoyoClinical Trial Unit (BCTU) of the Ifakara Health Institute(IHI). Volunteers were screened using predetermined inclu-sion and exclusion criteria based on clinical examinationsand laboratory tests. These included medical history andphysical examinations, and standard hematology, biochemis-try, malaria, human immunodeficiency virus, hepatitis B andC, and sickle cell tests. In addition subjects were screenedfor a-thalassemia. In the initial screening a-thalassemia traitwas an exclusion criterion because of a theoretical concernthat these individuals might be less susceptible to Pf infec-tion. As screening progressed it became clear that a sub-stantial proportion of the local population was heterozygousfor a-thalassemia and it would be important to include thispopulation in the volunteer pool to understand if the hetero-zygous condition was more resistant to malaria infection byexperimental challenge and pose an obstacle that wouldneed to be addressed in future vaccine studies. Volunteerswho indicated that they had not had an episode of docu-mented malaria in the past 5 years were included. They alsohad malaria thick smears, and any subject who was positivewas excluded.Screening for a+ and a0 a-thalassemia caused by deletions.

One milliliter (1 mL) of venous blood was collected inEDTA tubes and stored at −80°C. The DNA was extractedfrom 100 mL of whole blood with ZR Genomic DNA-TissueMiniPrep (ZymoResearch, Irvine, CA) according to manu-facturer’s recommendations. We used primers to amplify thealpha 2 globin gene as a control and the 3.7 kb, 4.2 kb, and20.5 kb deletion junction fragments of the a-thalassemiavariants that could be easily identified by size as described.15

The multiplex polymerase chain reaction (PCR) primersused were a 2/3.7del F, 3.7del/20.5del R, a2 R, 4.2del F,4.2del R, and 20.5del F. The PCR reaction contained addi-tionally 1 + Q-solution 2.5 U HotStarTaq DNA poly-merase in supplied reaction buffer (Qiagen, Valencia, CA)and 100 ng of genomic DNA. Reactions were carried outon a thermal cycler (Gene Amp 2700, Applied Biosystems,Foster City, CA), with an initial 15-minute denaturation at96°C, 30 cycles of 98°C for 45 seconds, 60°C for 90 seconds,72°C for 135 seconds, and a final extension at 72°C for5 minutes. Following amplification, 10 mL of the product wereelectrophoresed through a 1.5% agarose gel with 0.6 mg/mLethidium bromide in 1 + TBE buffer first at 7 volts/cm for1 hour followed by 3 volts/cm for an additional 2 hours. Thegel was visualized on an UV transilluminator. The wild-typeaa/aa loci yielded a PCR product of 1,800 base pair (bp),whereas the 3.7 kb deletion, 4.2 kb deletion and 20.5 kbdeletion resulted in PCR products of 2,029 bp, 1,628 bp,and 1,007 bp, respectively.Screening for a+ and a0 a-thalassemia caused by non-

deletion mutations. The three exons of each alpha 2 andalpha 1 gene were amplified and sequenced to determine allnon-deletional mutations that cause a+ and a0 a-thalassemia.Each 25 uL reaction contained 0.5 mM of each primer hemalpha F, hem alpha 1 R, hem alpha 2 R, and also 1 +Q-solution2.5 U HotStarTaq DNA polymerase in supplied reactionbuffer (Qiagen) and 100 ng of genomic DNA. Reactionswere carried out on a thermal cycler (Gene Amp 2700,

Applied Biosystems), with an initial 15-minute denaturationat 95°C, 38 cycles of 95°C for 20 seconds, 60°C for 20 sec-onds, 72°C for 90 seconds, and a final extension at 72°Cfor 5 minutes. Following amplification, 5 mL of product waselectrophoresed through a 1.5% agarose gel with 0.6 mg/mLethidium bromide in 1 + TBE buffer at 7 volts/cm for 1 hour.The gel was visualized on an UV transilluminator. ResultingPCR products were purified with a QIAquick PCR Purifica-tion Kit (Qiagen) and sequenced on both strands with theBig Dye Terminator v3.1 sequencing kit on an ABI 3130XLsequencer (Applied Biosystems). The Sequence reads werealigned against reference genes for hemoglobin alpha 1and 2 (NCBI Gene ID: 3039 and 3040, GRCh37.p10) andpolymorphisms were identified according to human dbSNP(Build 137). Sequence analysis was done by the Geneious 6.1.5software package.All volunteers gave written informed consent before

screening and being enrolled in the study. The trial wasperformed in accordance with Good Clinical Practices, anInvestigational New Drug (IND) application filed with theU.S. Food and Drug Administration (US FDA) (IND 14267),and an Investigational Medical Product Dossier (IMPD) filedwith the Tanzanian Food and Drug Administration (TFDA).The protocol was approved by institutional review boards(IRBs) of the Ifakara Health Institute ((IHI/IRB/No25) andNational Institute for Medical Research Tanzania (NIMR/HQ/R.8a/Vol.IX/1217), and the Ethikkommission beider Basel(EKBB), Basel, Switzerland (EKBB 319/11). The protocolwas also approved by TFDA (Ref. No. CE.57/180/04A/50), andthe trial was registered at ClinicalTrials.gov (NCT01540903).Intervention and randomization. The intervention mate-

rial was aseptic, purified, cryopreserved PfSPZ (PfSPZ Chal-lenge) isolated from salivary glands of aseptically rearedmosquitoes.16,17 Details regarding the production, cryopres-ervation quality control, potency, and CHMI have beendescribed.11–13,18,19 The lot of PfSPZ Challenge used in thisstudy had been manufactured and then cryopreserved inliquid nitrogen vapor phase (LNVP) 12 months beforeadministration. It was a different lot than the lot used inthe Netherlands12; however, it was the same lot that wasused in Oxford in which 5 of 6 volunteers who received2,500 PfSPZ ID and 6 of 6 volunteers who received25,000 PfSPZ IM developed malaria.13 The results of thesporozoite membrane integrity (viability) and 6-day hepa-tocyte potency assay (potency) were similar to those forthe previous trials (Supplemental Table 1).12,13

Thirty eligible volunteers were randomly allocated tothe experimental (PfSPZ Challenge) or control (normalsaline) groups. Twenty-four volunteers received PfSPZ Chal-lenge and six received normal saline (BD Medical/Surgical, BDPre-filled Normal Saline Flush Syringe, Columbus, Nebraska).The volunteers and clinicians were blinded as to whether thevolunteers received PfSPZ Challenge or normal saline.CHMI. Administration of PfSPZ Challenge and immedi-

ate follow-up. PfSPZ Challenge was administered ID intwo different dose groups. Twelve volunteers were inocu-lated with 10,000 PfSPZ ID in two injection sites, one50 mL injection in each deltoid, each injection containing5,000 PfSPZ. Twelve volunteers were inoculated with25,000 PfSPZ ID in four injection sites, two 10 mL injectionsin each deltoid, each injection containing 6,250 PfSPZ. Threecontrol volunteers were assigned to each of the two dose

472 SHEKALAGHE AND OTHERS

groups and were inoculated with normal saline in the sameway as those in that experimental group.Immediately before use, a vial of PfSPZ Challenge was

thawed and diluted with phosphate buffered saline con-taining human serum albumin in an aseptic environment. Itwas then injected by a blinded nurse within 30 minutesof thawing. After injection, volunteers were observed in theinjection room for at least 5 minutes and thereafter wereescorted by a nurse to the ward.Diagnosis of malaria. Thick blood smears were obtained

every 12 hours on Days 5 through 14 after injection withsaline or PfSPZ Challenge and daily on Days 15 through21 until positive or until Day 21. After initiation of treat-ment of a positive thick smear, thick smears were assesseduntil three consecutive daily smears were negative after treat-ment. Thick smears were also assessed on Day 28 after CHMI.Slide preparation and reading was performed following

a standard procedure. In brief, 10 mL of blood was placeduniformly on a 10 mm + 20 mm area of the slide, air dried,and stained with Giemsa, pH 7.2. Microscopes were cali-brated and the number of passes/fields required to read0.5 mL blood was determined. This amount of blood wasassessed for the primary reads to determine if the slidewas negative. If the volunteer was symptomatic, double thisamount of blood was read. A blood smear was declaredpositive when one reader saw two parasites in 0.5 mL ofblood and the presence of parasites was independentlyconfirmed by a second reader. The pre-patent period wasdefined as the period between inoculation of PfSPZ Chal-lenge and appearance of the first positive blood smear.Retrospectively, parasitemias were also determined by quan-titative polymerase chain reaction (qPCR) performed on allsamples collected after CHMI, as previously described.20

Treatment of malaria. Those who became smear positivewere treated with a standard 3-day regimen of artemether/lumefantrine (Coartem), and were discharged after three con-secutive negative smear results. Those who did not becomepositive during the first 21 days after CHMI were dischargedand returned on Day 28. On this day, the study was unblindedand those who had received PfSPZ Challenge and had notdeveloped infection were treated with artemether/lumefantrine(Coartem) irrespective of the blood smear results. All volun-teers were seen on Days 56 and 168.Assessment of adverse events. The volunteers were observed

in the ward for 24 hours after administration of PfSPZ Chal-lenge and discharged to home. They were given diaries andthermometers for recording of adverse events and tempera-tures. Volunteers returned on Day 5 after administration ofPfSPZ Challenge for admission to the ward for assessmentof safety and diagnosis and treatment of malaria.During the period of follow-up all symptoms and signs

(solicited and unsolicited) were recorded and graded bythe attending physician as follows: mild (easily tolerated),moderate (interferes with normal activity), or severe (pre-vents normal activity). Axillary temperature was recorded asgrade 1 (> 37.5–38.0°C), grade 2 (> 38.0–39.0°C), or grade 3(> 39.0°C). Hematological and biochemical parameterswere assessed minimally on Days 5, 9, 12, 28, 56, and 168after inoculation of PfSPZ Challenge, and on the day ofparasite positivity (day of initiating treatment). For thoseindividuals who did not become positive by thick blood smear,these assays were also conducted on Days 15, 18, and 21 after

inoculation of PfSPZ Challenge. Results were graded accord-ing to a predetermined table (Supplemental Table 2 adaptedfrom FDA guidelines, http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm091977.pdf). Adverse events weredivided into those that occurred during the 5 days after inocu-lation of PfSPZ Challenge, and were attributed to the admin-istration of the study product, and those that occurred fromDay 6 onward, and were attributed to Pf infection (malaria).The possibility of cardiac damage was assessed, because

a cardiac-related serious adverse event (SAE) had beenreported in 2007 in the Netherlands in a volunteer who wasimmunized with an experimental Pf subunit vaccine, under-went CHMI by exposure to the bites of five Pf-infectedmosquitoes, developed malaria, and was treated with an anti-malarial.21 In case symptoms or signs that could be related to acardiac event developed during the study, blood was collectedat baseline to be able to determine if there was any differencein the results of assays used to assess cardiac damage (e.g.,troponins) before the trial began and at the time of suchan event. We note that after our clinical trial in Tanzania,another cardiac event occurred in a volunteer in a PfSPZ-CVac vaccine trial in the Netherlands after CHMI by mos-quito bite, diagnosis of malaria, and initiation of treatment.22

Genotyping of parasites. At the time of diagnosis andbefore treatment 4 mL of blood were collected for geno-typing by microsatellite analysis to determine if the para-sites were derived from PfSPZ Challenge (Pf NF54) or froma naturally acquired infection. The DNA was isolated fromblood specimens using the QIAamp DNA Blood Midi Kit(Qiagen). Microsatellite markers Poly alpha, PfPK2, TA81,ARA2, TA87, and TA40 were amplified using hemi-nestedPCR.23,24 Capillary electrophoresis was performed using anApplied Biosystems 3730XL 96-capillary DNA sequencerand software. Capillary electrophoresis output files wereanalyzed using Genemapper 4.0 (Applied Biosystems). Geno-mic control strains 3D7 and HB3 (ATCC-MR4, ManassasVA) were included to determine characteristic peak mor-phology for each microsatellite locus and control for slightvariations among runs. Microsatellite peaks above thethreshold of 100 relative fluorescent units exhibiting charac-teristic morphology were scored. Peak sizes were deter-mined by manual inspection of each electropherogram andthen normalized against the Pf 3D7 control. Normalized peaksizes were compared with those observed in Pf NF54.Statistical analysis. Data analysis was performed using

SAS software version 9.3 (SAS Institute Inc., Cary, NC).Descriptive statistics were assessed, specifically the geometricmean, for the parasitemia results. Thick blood smear andqPCR results were compared between the 10,000 and 25,000PfSPZ groups using a non-parametric test (Wilcoxon rank-sum test, two-tailed). The effects of a-thalassemia hetero-zygosity on parasitemia were compared within each group bynon-parametric tests (Wilcoxon rank-sum test, two-tailed).Proportions were compared using c2 test (two-tailed).

RESULTS

Volunteers. In total 323 volunteers were recruited fromhigher learning institutions during the first informationmeeting. Out of these, 30 met eligibility criteria and wereenrolled and randomized to the 10,000 PfSPZ dose (N = 12),

INJECTION OF TANZANIANS WITH P. FALCIPARUM SPOROZOITES 473

25,000 PfSPZ dose (N = 12), or control (N = 6) groups(Figure 1 and Table 1). In groups 1, 2, and the controlgroup, 7, 1, and 2 volunteers were heterozygous for thea-thalassemia trait, respectively.Parasitemia. Thick blood smear. Eleven volunteers who

received 10,000 PfSPZ (Table 2A) and 10 volunteers whoreceived 25,000 PfSPZ (Table 2B) developed parasitemiadetected by thick blood smear and confirmed by qPCR.One of the other two subjects in the 25,000 PfSPZ groupwas treated for a false positive thick blood smear onDay 11 (qPCR was negative), and was eliminated from theanalysis. The second other volunteer in the 25,000 PfSPZgroup was treated for a false positive thick blood smear onDay 19; qPCR was negative throughout for this volunteer,and this volunteer was considered to have not developed

parasitemia as no subject in the study developed parasitemiaby qPCR after Day 16, and this volunteer was followed byqPCR through Day 19. Thus, 11 of 12 volunteers in the10,000 PfSPZ group and 10 of 11 evaluable volunteers in the25,000 PfSPZ group developed Pf parasitemia after ID injec-tion of PfSPZ Challenge. Volunteers in the 10,000 PfSPZgroup had a significantly different pre-patent period than inthe 25,000 PfSPZ group (geometric mean [GM] of 15.4 and13.5 days, Wilcoxon, P = 0.023). The GM parasite densitieswere 8.9 and 7.0 parasites/mL blood, respectively, at the timeof first thick smear positivity.qPCR. Parasitemia was determined by qPCR on all sam-

ples collected after CHMI. The qPCR was performed retro-spectively, after volunteers had been diagnosed and treated.The sensitivity of qPCR was considered to be 20 parasites/mLof blood. The qPCRs were first positive 9.0 to 16.0 days afterinoculation of PfSPZ Challenge (Table 2). Consistent withthick blood smear results, the volunteers in the 10,000 PfSPZgroup had a longer time to positive qPCR than those in the25,000 PfSPZ group (GM of 12.6 and 11.1 days, respectively),however the difference did not reach the level of statisticalsignificance (Wilcoxon, P = 0.076). The GM parasite densitieswere 0.11 and 0.16 parasites/mL blood (110 and 160 parasites/mL blood), respectively, at the time of first qPCR positivity.The GM parasite densities by qPCR at the time of thick smeardiagnosis in the two groups were 4.1 and 1.6 parasites/mL,respectively. qPCR was negative throughout the 21-day follow-up for the slide-negative volunteer who received 10,000 PfSPZ,and through Day 19 for the subject who received 25,000 PfSPZand was treated on Day 19. It was also negative in all normalsaline controls, except for one specimen on Day 19, whichwas determined to be caused by mislabeling of a specimenfrom a PfSPZ Challenge subject. Parasite growth was cyclicaland similar in both dose groups (Figure 2). Using a pre-viously described method,25 the parasite multiplication ratein the bloodstream could not be determined with confidencebecause of the high variability of the amplification dynamicsamong individual subjects. However, there did not appear to

Figure 1. Flow chart of recruitment and study design.

Table 1

Volunteer characteristics*Control

(normal saline)Group 1

(10,000 PfSPZ)Group 2

(25,000 PfSPZ)

N = 6 N = 12 N = 12

SexMale 6 12 12

Age at screening (years)Mean ± SD 25.7 ± 3.0 25.9 ± 1.6 24.6 ± 2.3Median 25.2 25.5 24.5Min, max 21.5, 30.9 24.3, 30.5 20.7, 27.2

BMI (kg/m2)Mean ± SD 20.7 ± 2.4 21.7 ± 3 20.5 ± 2.3Median 19.3 20.7 19.7Min, max 19, 24 18.4, 27.9 17.3, 25.4

Height (cm)Mean ± SD 168 ± 4.3 168.5 ± 8.2 171.7 ± 5.6Median 166 169 172Min, max 164, 174 158, 182 162, 179

Weight (kg)Mean ± SD 58.4 ± 6.3 61.7 ± 9.8 60.5 ± 8.8Median 58.3 60.8 60.5Min, max 51, 66 47, 79 45.5, 81.5

*BMI = body mass index.

474 SHEKALAGHE AND OTHERS

be a significant difference between the replication rate in theTanzanians and the replication rate in the Dutch subjects.12

Parasite genotypes. Parasites from the positive volunteerswere genotyped at six polymorphic microsatellite loci to con-firm that they were from PfSPZ Challenge (Pf NF54) and notacquired by natural exposure to infected mosquitoes in theBagamoyo area. Parasites from all 21 volunteers had identi-cal microsatellite signatures to Pf NF54 (Table 3), indicat-ing that they were infected with the challenge strain andnot locally transmitted Pf parasites. Later, on Days 67 and89 after CHMI, two volunteers developed Pf malaria. Theseinfections were also genotyped and both carried non-PfNF54 alleles at all six microsatellite loci (Table 3), indicat-ing that they were infected with naturally acquired, locallytransmitted Pf parasites.Effect of a-thalassemia heterozygosity on parasitemia. The

GM parasite density for volunteers who were heterozygousfor a-thalassemia and those who were non-a-thalassemicin the 10,000 PfSPZ were not significantly different at thetime of detection of parasites by microscopy, 11.1 versus6.9 Pf/mL blood, respectively (Wilcoxon, P = 0.5498) (Table 4).The GM time to blood smear positivity (pre-patent period)was also not significantly different between a-thalassemiaheterozygous and non- a-thalassemia volunteers, 15.4 and15.4 days, respectively, in the 10,000 PfSPZ group (Wilcoxon,P = 0.7922). In the 25,000 PfSPZ group, the parasite den-

sities at the time of detection of parasites by microscopywas 4.0 parasites/mL blood in the one a-thalassemia hetero-zygous volunteer and 7.4 parasites/mL blood (GM) in non-a-thalassemia volunteers. The GM time to blood slide positivityin the a-thalassemia heterozygous subjects was not sig-nificantly different from the non-a-thalassemia subjects,12.7 versus 13.6 days (Wilcoxon, P = 0.7000) (Table 4).Adverse events (AEs). Clinical adverse events.A summary

of the number of volunteers in each group with AEs duringthe 28 days after injection of normal saline or PfSPZ Chal-lenge is shown in Table 5. The occurrence of AEs wassimilar in all groups. Most AEs recorded after administra-tion of PfSPZ were consistent with symptoms and/or signsassociated with clinical malaria. Table 6 provides the totalnumber of AEs that occurred during Days 0–28 brokendown by Grade (1–3); 75% were Grade 1. Table 7 delineatesthe specific solicited and unsolicited AEs that occurredduring Days 0–28 after injection of normal saline or PfSPZChallenge. There were 63 AEs in groups 2 (10,000 PfSPZ)and 3 (25,000 PfSPZ) and only nine of the 63 AEs (14%)occurred during the first 5 days after injection of PfSPZChallenge. There were no acute systemic allergic reactionsafter the injection. The most common study-related eventswere headache, malaise, fatigue, and arthralgia, all symp-toms consistent with malaria. Surprisingly, saline controlsubjects who did not have malaria had these symptoms

Table 2

A. Thick smear and qPCR results, group 1 (10,000 PfSPZ)

Volunteer code

Thick smear qPCR

Pre-patentperiod (day)

Parasite densityat diagnosis (Pf/mL)

qPCRpositive (day)

Parasite density atfirst day positive (Pf/mL)

Parasite density by qPCR at timeof diagnosis by thick smear (Pf/mL)

10002-20 18.6 5.0 16.0 0.24 0.0110023-20 18.7 15.0 15.0 0.12 13.0030035-20 18.7 5.0 14.0 0.04 11.0040010-20 N/A N/A N/A N/A N/A50041-20 14.6 4.0 13.0 0.36 3.0060008-20 12.8 4.0 10.5 0.11 8.0060026-20 12.7 11.0 11.5 0.21 9.0070001-20 14.2 80.0 11.0 0.21 23.0070014-20 15.8 21.0 12.5 0.03 83.0070031-20 14.2 11.0 11.0 0.05 17.0070044-20 17.6 6.0 15.0 0.07 6.0090047-20 13.7 4.0 11.0 0.09 0.10Geom. mean 15.4 8.9 12.6 0.11 4.10No. of positives 11/12 11/12

B. Thick smear and qPCR results, group 2 (25,000 PfSPZ)

20056-20 18.7 5 16.0 0.13 14.0020064-20 11.1 7 9.0 0.07 5.0020070-20 12.6 13 9.5 0.07 5.0030053-20 13.7 4 12.0 0.17 0.1730060-20 13.5 7 11.0 1.00 0.4040055-20 N/A N/A* N/A N/A N/A40068-20 13.4 7 11.0 0.26 2.0050050-20 12.7 7 10.0 0.04 10.0050057-20 N/A N/A† N/A N/A N/A60051-20 12.7 4 11.0 0.13 1.0060072-20 14.0 7 11.5 0.17 1.0080058-20 13.7 15 11.0 0.36 0.12Geom. mean 13.5 7 11.1 0.16 1.60No. of positives 10/11 10/11

*This volunteer was treated on Day 19 as a result of reporting of a positive thick blood smear. Review of this thick blood smear indicated that it was negative, and all qPCR results onthis volunteer were negative. Thus, this volunteer was considered as negative for Plasmodium falciparum infection for subsequent analyses, because no volunteers first became qPCR positiveon Day 19 or later.†This volunteer was treated on Day 11 as a result of reporting of a positive thick blood smear. Review of this thick blood smear indicated it was negative, and all qPCR results on

this volunteer were negative. Thus, it is not known if this volunteer would have developed Plasmodium falciparum infection, and this volunteer has been excluded from the analysis. Thus,11 volunteers were considered to be in this group, and 10 were documented to have developed P. falciparum parasitemia.

INJECTION OF TANZANIANS WITH P. FALCIPARUM SPOROZOITES 475

at a frequency similar to that of subjects who receivedPfSPZ Challenge.Clinical serious adverse events (SAEs)—all unrelated.

There were two subjects who had unrelated SAEs in thisstudy. The first SAE was in a 26-year-old volunteer whowas diagnosed as having gastroenteritis/dysentery of grade 3severity at the time of hospitalization, which was 33 dayspost administration of PfSPZ Challenge. The second SAEoccurred in a 22-year-old volunteer who was admitted tothe hospital and diagnosed with severe malaria (axillarytemperature > 40°C, altered mental status, and smear posi-tive) on day 89 after administration of PfSPZ Challenge (the

volunteer was previously treated and successfully cleared ofparasites on Day 14 post inoculation of PfSPZ Challenge).Parasites taken from this volunteer at the time of diagno-sis of severe malaria were genotyped using microsatellitemarkers, and these parasites were shown not to be the PfNF54 parasites in PfSPZ Challenge (see above, and Table 3).Both volunteers with SAEs recovered uneventfully.Laboratory test abnormalities. Serum chemistries (glucose,

creatinine, aspartate aminotransferase [AST], alanine amino-transferase [ALT], and bilirubin) and hematological parame-ters (e.g., hemoglobin, white blood cell count, platelet count)were assessed beginning on Day 5 after inoculation of normal

Figure 2. Parasite density as measured by qPCR in the 10,000 (A) and 25,000 (B) PfSPZ dose groups. Panels A (N = 11) and B (N = 10)show individual and geometric mean parasite density (parasites/mL) of positive volunteers from day of inoculation through last day ofpositivity after initiation of treatment. Panel C shows an overlay of geometric mean parasite densities with confidence intervals (95%) ofpositive volunteers in each group.* *For Panel B, the geometric mean was calculated excluding the single volunteer who first became positiveon Day 16. This figure appears in color at www.ajtmh.org.

476 SHEKALAGHE AND OTHERS

saline or PfSPZ Challenge. When assessed on Day 5 afterinoculation of normal saline and PfSPZ Challenge, labora-tory abnormalities were infrequent and self-limited and simi-lar in incidence rate among all three groups (SupplementalTable 3). From Day 6 to Day 28 post inoculation the inci-

dence rate of abnormalities was slightly increased, but theincidence rate of abnormalities was similar in normal salinecontrols and those who received PfSPZ Challenge (Supple-mental Table 3).

DISCUSSION

In this study, we showed for the first time that inoculationof healthy, young adult, African males with aseptic, purified,cryopreserved P. falciparum sporozoites, a product calledPfSPZ Challenge, was safe, well tolerated, and infective. Infact, the infection rates in the Tanzanians were as good, ifnot better than the infection rates in young adult Dutchwith no previous exposure to malaria.12

Eleven of 12 subjects who received 10,000 PfSPZ ID intwo divided 50 mL doses and 10 of 11 who received 25,000PfSPZ ID in four divided 10 mL doses became parasitemic.In the Netherlands, 5 of 6 who received 10,000 and 5 of 6who received 25,000 PfSPZ ID in two divided 50 mL dosesbecame parasitemic.However, the GM pre-patent period for the Tanzanians

who received the 10,000 PfSPZ dosage regimen was15.4 days, and the GM pre-patent period for the Dutchwas 12.6 days (P = 0.0192, Wilcoxon 2-tailed). There aremultiple possible explanations for the prolonged pre-patentperiod in the Tanzanians. One is that naturally acquiredimmunity reduced the number of parasites that invaded orfully developed in the liver, thereby reducing the numbers ofparasites that were released from the liver and prolongingthe pre-patent period. Another possibility is that the repli-cation rate in the blood was reduced as a result of natu-rally acquired immunity or innate resistance, and that thisreduced replication prolonged the pre-patent period. Unfor-tunately, the variability in the qPCR results did not allow fordefinitively determining if this was the case, but it appeared

Table 3

Microsatellite genotypes of Pf infections in volunteers after PfSPZChallenge

Strain orsample ID

Microsatellite Loci*

TA81 Poly a PfPK2 TA40 ARA2 TA87

DNA controls HB3 131 178 190 205 61 1033D7 122 148 166 220 64 94NF54 122 148 166 220 64 94

Post-CHMI infections 7059065 122 148 166 220 64 948059066 122 148 166 220 64 949059067 122 148 166 220 64 942058376 122 148 166 220 64 943059070 122 148 166 220 64 944059071 122 148 166 220 64 945059072 122 148 166 220 64 –†6059073 122 148 166 220 64 947059074 122 148 166 220 64 948059075 122 148 166 220 64 949059076 122 148 166 220 64 941059077 122 148 166 220 64 942059078 122 148 166 220 64 943059079 122 148 166 220 64 944059080 122 148 166 220 64 945059081 122 148 166 220 64 943059088 122 148 166 220 64 947059083 122 148 166 220 64 948059084 122 148 166 220 64 949059085 122 148 166 220 64 946059082 122 148 166 220 64 94

Subsequent infections 4155794 118 155 157 228 59 1037060514 118 155 163 217 87 88

Gray shading indicates microsatellite alleles shared with NF54.*Numbers represent microsatellite sizes in nucleotide base pairs, as determined by

capillary electrophoresis.†Did not amplify.

Table 4

Pre-patent periods and parasite densities by microscopy by a-thalassemia status

Heterozygous Normal

Volunteer IDPre-patent

period (Days)Parasite densityat diagnosis Volunteer ID

Pre-patentperiod (Days)

Parasite densityat diagnosis

Group 1*90047-20 13.74 4.0 50041-20 14.64 4.070001-20 14.11 80.0 60008-20 12.75 4.060026-20 12.71 11.0 70044-20 17.63 6.070014-20 15.76 21.0 70031-20 14.16 11.010002-20 18.64 5.0 10023-20 18.73 15.030035-20 18.66 5.0

Geom. mean 15.43 11.1 15.42 6.9No. positive 6/7 5/5

Group 2*60051-20 12.72 4.0 30053-20 13.72 4.0

80058-20 13.68 15.020056-20 18.67 5.040068-20 13.44 7.030060-20 13.47 7.050050-20 12.72 7.060072-20 14.04 7.020070-20 12.62 13.020064-20 11.05 7.0

Geom. mean 12.72 4.0 13.59 7.4No. positive 1/1 9/9

*The proportion of volunteers who were heterozygous for a-thalassemia trait was higher in the 10,000 PfSPZ group than in the 25,000 PfSPZ group (chi-squared, 2-tailed, P = 0.011). There wereno statistically significant differences in infection rates, parasitemia, or parasites/mL blood between subjects heterozygous for a-thalassemia trait, and those who were not.

INJECTION OF TANZANIANS WITH P. FALCIPARUM SPOROZOITES 477

that the replication rate was similar to the 11.5-fold replica-tion rate every 48 hours seen in the Dutch volunteers.12

Thus, we do not know why the pre-patent period waslonger in the Tanzanians. However, because the replica-tion rates seemed to be similar in the Dutch and Tanzaniansubjects, we think that there may have been fewer mero-zoites released from the livers of the Tanzanians.Interestingly, the GM pre-patent period in the group that

received 25,000 PfSPZ was significantly different (13.5 days)than was the pre-patent period in the group that received10,000 PfSPZ (15.4 days) (P = 0.023). This 2-day differencecan only partially be accounted for by increasing the numbersof PfSPZ by 2.5-fold. It is likely that more efficient adminis-tration of the PfSPZ contributed to the delay by increasingthe number of sites of administration from two to four anddecreasing the volume of the injections from 50 to 10 mL, aswe have seen in mice.14

Hemoglobinopathies, disorders of hemoglobin structure,and production, are one of the most common monogenic dis-orders in humans. The a-thalassemia is a hemoglobinopathyresulting from deletion of one (−a) or both (–) a genes fromchromosome 16. Its wide distribution in populations livingin places like Tanzania, where malaria has been or still ispresent, has been hypothesized to result from protectionagainst severe or lethal malaria.26 This study was notdesigned (i.e. powered) to be able to assess the differences ininfection rates, pre-patent periods, and parasite densitiesbetween individuals with and without a-thalassemia trait.Nonetheless, there was no indication of any differencesbetween those carrying this trait and those who did nothave it. This finding is consistent with a number of field

studies that showed that individuals who were heterozygousor homozygous for a-thalassemia had similar rates of asymp-tomatic parasitemia and similar manifestations of uncompli-cated malaria as non-thalassemic individuals.27

Administration of PfSPZ Challenge was extremely welltolerated. There were no serious adverse events. In fact,the incidence rate of AEs was the same in the control groupthat received normal saline as it was in the groups thatreceived PfSPZ Challenge (Tables 5–7). Very few of theAEs occurred during the first 5 days after inoculation ofPfSPZ Challenge, and thus very few were attributed to theinjection of PfSPZ Challenge (Table 7). Most occurredduring the period when the volunteers were being diag-nosed with Pf parasitemia and being treated. However, evenduring this period the incidence rate was similar in thecontrol and experimental groups. One of the striking find-ings was how few symptoms and signs attributable to Pfparasitemia were experienced by the subjects who developedmalaria. In the Dutch study 9 of 18 volunteers reported fever,whereas only 2 of 24 (P = 0.004, Fisher’s exact test, 2-tailed)reported fever in this study. In the Netherlands 5 of18 had chills, whereas in this study only 1 of 24 had chills(P = 0.068).12 We assume this low level of symptoms andsigns was attributable to naturally acquired or innate immu-nity, which ameliorated clinical manifestations.A panel of six polymorphic microsatellite loci was success-

fully used to identify challenge strain (Pf NF54) infectionsand differentiate them from naturally acquired P. falciparum

parasites circulating in Bagamoyo. This technique has beenpreviously shown to be highly effective at differentiatinggenetic variants of P. falciparum within countries and across

Table 5

Number of volunteers with clinical adverse events, Days 0–28 postinjection*

Salinecontrol(N = 6)

Group 1(10,000 PfSPZ)

(N = 12)

Group 2(25,000 PfSPZ)

(N = 12)

Adverse events(no. of volunteers, %)

n (%) n (%) n (%)

Any adverse event 6 (100) 9 (75) 9 (75)Serious adverse event 0 0 0Solicited adverse event 6 (100) 9 (75) 9 (75)Unsolicited adverse event 2 (33) 6 (50) 2 (17)

*A volunteer was counted at most once within each event type.

Table 6

Number of clinical adverse events, Days 0–28 post-injection

Salinecontrol(N = 6)

Group 1(10,000 PfSPZ)

(N = 12)

Group 2(25,000 PfSPZ)

(N = 12)

Total number of AEs 22 42 42Total solicited AEsGrade 1 14 30 34Grade 2 4 2 6Grade 3 2* 1† 0

Total unsolicited AEsGrade 1 2 8 1Grade 2 0 0 1Grade 3 0 1‡ 0

Total SAEs 0 0 0

An adverse event (AE) was recorded more than once if it resolved and subsequentlyreappeared during the 28-day interval.*Severe rash and pruritis (each grade 3) in a volunteer beginning 10 days post-injection.†Severe headache attributable to controlled human malaria infection (CHMI).‡Pharyngitis, etiology unclear, resolved after 3 days.SAE = serious adverse event.

Table 7

Specific solicited and unsolicited adverse events, Days 0–28 post–injection*

Adverse event

Salinecontrol

Group 110,000 PfSPZ

Group 225,000 PfSPZ

N = 6 N = 12 N = 12

Total AEs 21 (3) 30 (3) 34 (6)Fever 0 0 2Headache 6 (1) 7 7 (1)Malaise 1 5 (1) 4Fatigue 3 (1) 5 6 (2)Myalgia 3 2 1Arthralgia 3 2 4Nausea and/or vomiting 0 0 0Chills 0 0 1Diarrhea 0 0 0Constipation 0 1 0Abdominal pain 1 0 1 (1)Chest pain/discomfort 0 1 0Palpitations 0 0 0Shortness of breath 0 0 0Dizziness 1 0 0Erythema, swelling,or pruritis at injection site

0 0 3 (2)

Rash, remote to injection site 1 0 0Itching throat 0 0 1Loss of appetite 0 3 (1) 1Pain on swallowing/pharyngitis 1 2 0Back pain 0 1 (1) 0Neck pain 0 1 0Elevated axillary temp. 0 0 3

*A volunteer was counted at most once within each event type, even if the AE clearedand reappeared. All AEs were considered either possibly or probably related to the injec-tion of study product or malaria. Numbers in parentheses occurred within the first 5 daysafter injection of study product.

478 SHEKALAGHE AND OTHERS

larger geographic regions.28–32 Here, we show that the assayis also sensitive enough to generate reliable multi-locus geno-types from infected blood with parasite densities as low asfive parasites/mL blood. Although microsatellites are usefulin identifying the infecting strain, they do not provide infor-mation on genetic regions of vaccine-induced selection orescape following heterologous challenge. Future studies will

include genome-wide characterization of breakthrough infec-tions in PfSPZ Vaccine11,18,19 trials using DNA microarraysand genome sequencing to assess cross-strain protection andidentify loci under vaccine-induced immune selection. Thisapproach may help to pinpoint the genetic regions encod-ing the key antigens responsible for driving strain-specific

immune responses and inform the development of next-generation multivalent whole organism vaccines in the eventthat efficacy is found to be strain-specific.In conclusion, we have established the foundation for

using CHMI with PfSPZ Challenge in Africans to establish

the efficacy of new interventions against malaria and to studythe mechanisms of protection conferred by hemoglobinopathies,glucose 6 phosphate dehydrogenase deficiencies, and innateand acquired immunity to malaria in settings where malaria

is endemic. Improvements in administration, such as directvenous inoculation (DVI) (Mordmueller B, submitted), willsoon be used in Bagamoyo in CHMI studies to assess theprotective efficacy of the PfSPZ Vaccine19 in Tanzanians.

Received February 26, 2014. Accepted for publication May 25, 2014.

Published online July 28, 2014.

Note: Supplemental tables appear at www.ajtmh.org.

Acknowledgments: We thank all volunteers and staff of the IfakaraHealth Institute, Bagamoyo Research, and Training Centre, SanariaInc., and Swiss Tropical and Public Health Institute who contributedso much to this project. Special thanks to Yonas Abebe, AlexanderHoffman, Thomas L. Richie, and Robert C. Thompson of Sanariafor editorial, legal, and administrative support, and the Sanariamanufacturing, quality control, regulatory, and clinical teams. Wethank Rose Minja, Hery Mwandolela, and Ally Mtoro for safetyreview of volunteers during the trial and Pharmacist Abel Mdemufor verification of formulation. We appreciate the advice of BarneyGraham, David Diemert, and Zulfiqarali Premji, members of theSafety Monitoring Committee.

Financial support: The Tanzanian Commission on Science and Tech-nology (COSTECH), the Ifakara Health Institute, and the SwissTropical Public Health Institute provided funding for this clinicaltrial. The development, manufacturing, and quality control releaseand stability studies of PfSPZ Challenge was supported in part by anNIAID Small Business Innovation Research grant, 5R44AI058375.

Disclosure: Sanaria Inc. manufactured PfSPZ Challenge, and ProteinPotential LLC is affiliated with Sanaria. Thus, all authors associatedwith Sanaria or Protein Potential have potential conflicts of interest.There are no other conflicts of interest.

Authors’ addresses: Seif Shekalaghe, Mastidia Rutaihwa, MwajumaChemba, Maximillian Mpina, Omar Ali Juma, Beatus Simon, OmarLweno, Paul Mutani, Ali Hamad, Ali Mohammed, Alwisa Urassa,Tutu Mzee, and Salim Abdullah, Ifakara Health Institute, BagamoyoResearch and Training Centre, Bagamoyo, Tanzania, E-mails:sshekalaghe@ihi.or.tz, mrutaihwa@ihi.or.tz, mchemba@ihi.or.tz,Ojuma@ihi.or.tz, bbongole@ihi.or.tz, mmpina@ihi.or.tz, olweno@ihi.or.tz, pmutani@ihi.or.tz, ahamad@ihi.or.tz, amohammed@ihi.or.tz,aurassa@ihi.or.tz, tmzee@ihi.or.tz, and sabdulla@ihi.or.tz. Peter F.Billingsley, Eric R. James, Anusha Gunasekera, AnitaManoj, ElizabethSavarino, Debbie Padilla, Adam Ruben, and Stephen L. Hoffman,Sanaria Inc., Rockville, MD, E-mails: pbillingsley@sanaria.com,ejames@sanaria.com, agunasekera@sanaria.com, amanoj@sanaria.com, esaverino@sanaria.com, dpadilla@sanaria.com, aruben@sanaria

.com, and slhoffman@sanaria.com. Claudia A. Daubenberger, TobiasSchindler, and Maximillian Mpina, Department of Medical Parasi-tology and Infection Biology, Swiss Tropical and Public Health Insti-tute, Basel, Switzerland, and University of Basel, Basel, Switzerland,E-mails: Claudia.Daubenberger@unibas.ch, Tobias.Schindler@stud.unibas.ch, and Max.Mpina@hotmail.com. Eric Huber, Departmentof Medicines Research, Swiss Tropical and Public Health Institute,Basel, Switzerland, E-mail: Eric.Huber@unibas.ch. L. W. PrestonChurch, Hospital Epidemiologist and Head Division of InfectiousDiseases VA Hospital, Charleston, SC, E-mail: lwpchurch@sanaria.com. Cornelus C. Hermsen and Robert W. Sauerwein, Radboud Uni-versity Nijmegen Medical Center, Department of Medical Micro-biology, Nijmegen, The Netherlands, E-mails: R.Hermsen@ncmls.ru.nland R.Sauerwein@mmb.umcn.nl. Christopher Plowe and MeeraVenkatesan, Howard Hughes Medical Institute and University ofMaryland Center for Vaccine Development, Baltimore, MD, E-mails:cplowe@medicine.umaryland.edu and mvenkate@medicine.umaryland.edu. Philip Sasi, Muhimbili University of Health and Allied Sci-ences, Dar es Salaam, Tanzania, E-mail: philip.sasi@gmail.com. B.Kim Lee Sim, Sanaria Inc., and Protein Potential LLC, Rockville,MD, E-mail: ksim@protpot.com. Marcel Tanner, Department of Epi-demiology and Public Health, Swiss Tropical and Public Health Insti-tute, Basel, Switzerland, and University of Basel, Basel, Switzerland,E-mail: Marcel.Tanner@unibas.ch.

This is an open-access article distributed under the terms of theCreative Commons Attribution License, which permits unrestricteduse, distribution, and reproduction in any medium, provided theoriginal author and source are credited.

REFERENCES

1. Mayne B, 1933. The injection of mosquito sporozoites in malariatherapy. Public Health Rep 48: 909–913.

2. Mayne B, Young M, 1941. The technique of induced malariaas used in the South Carolina State Hospital. J Vener Dis Inf22: 271–276.

3. Boyd MF, 1949. Malariology: A Comprehensive Survey of AllAspects of This Group of Diseases from a Global Standpoint.Philadelphia, PA: W. B. Saunders.

4. Ifediba T, Vanderberg JP, 1981. Complete in vitro maturationof Plasmodium falciparum gametocytes. Nature 294: 364–366.

5. Campbell CC, Collins WE, Nguyen Dinh P, Barber A, BrodersonJR, 1982. Plasmodium falciparum gametocytes from culturein vitro develop to sporozoites that are infectious to primates.Science 217: 1048–1050.

6. Chulay JD, Schneider I, Cosgriff TM, Hoffman SL, Ballou WR,Quakyi IA, Carter R, Trosper JH, Hockmeyer WT, 1986.Malaria transmitted to humans by mosquitoes infected fromcultured Plasmodium falciparum. Am J Trop Med Hyg 35:66–68.

7. Church LW, Le TP, Bryan JP, Gordon DM, Edelman R, FriesL, Davis JR, Herrington DA, Clyde DF, Shmuklarsky MJ,Schneider I, McGovern TW, Chulay JD, Ballou WR,Hoffman SL, 1997. Clinical manifestations of Plasmodiumfalciparum malaria experimentally induced by mosquitochallenge. J Infect Dis 175: 915–920.

8. Epstein JE, Rao S, Williams F, Freilich D, Luke T, Sedegah M,de la Vega P, Sacci J, Richie TL, Hoffman SL, 2007. Safetyand clinical outcome of experimental challenge of humanvolunteers with Plasmodium falciparum-infected mosquitoes:an update. J Infect Dis 196: 145–154.

9. Roestenberg M, O’Hara GA, Duncan CJ, Epstein JE, EdwardsNJ, Scholzen A, van der Ven AJ, Hermsen CC, Hill AV,Sauerwein RW, 2012. Comparison of clinical and parasito-logical data from controlled human malaria infection trials.PLoS ONE 7: e38434.

10. Laurens MB, Duncan CJ, Epstein JE, Hill AV, Komisar JL,Lyke KE, Ockenhouse CF, Richie TL, Roestenberg M,Sauerwein RW, Spring MD, Talley AK, Moorthy VS, 2012.A consultation on the optimization of controlled humanmalaria infection by mosquito bite for evaluation of candi-date malaria vaccines. Vaccine 30: 5302–5304.

11. Hoffman SL, Billingsley P, James E, Richman A, Loyevsky M,Li T, Charkravarty S, Gunasekera A, Chattopadhyay R, Li

INJECTION OF TANZANIANS WITH P. FALCIPARUM SPOROZOITES 479

M, Stafford R, Ahumada A, Epstein JE, Sedegah M, ReyesS, Richie TL, Lyke KE, Edelman R, Laurens MB, PloweCV, Sim BKL, 2010. Development of a metabolically active,non-replicating sporozoite vaccine to prevent Plasmodiumfalciparum malaria. Hum Vaccin 6: 97–106.

12. Roestenberg M, Bijker EM, Sim BK, Billingsley PF, James ER,Bastiaens GJ, Teirlinck AC, Scholzen A, Teelen K, Arens T,van der Ven AJ, Gunasekera A, Chakravarty S, VelmuruganS, Hermsen CC, Sauerwein RW, Hoffman SL, 2013. Con-trolled human malaria infections by intradermal injectionof cryopreserved Plasmodium falciparum sporozoites. Am JTrop Med Hyg 88: 5–13.

13. Sheehy SH, Spencer AJ, Douglas AD, Sim BK, Longley RJ,Edwards NJ, Poulton ID, Kimani D, Williams AR, AnagnostouNA, Roberts R, Kerridge S, Voysey M, James ER, BillingsleyPF, Gunasekera A, Lawrie AM, Hoffman SL, Hill AV, 2013.Optimizing controlled human malaria infection studies usingcryopreserved parasites administered by needle and syringe.PLoS ONE 8: e65960.

14. Ploemen IH, Chakravarty S, van Gemert GJ, Annoura T, KhanSM, Janse CJ, Hermsen CC, Hoffman SL, Sauerwein RW,2013. Plasmodium liver load following parenteral sporozoiteadministration in rodents. Vaccine 31: 3410–3416.

15. Tan AS, Quah TC, Low PS, Chong SS, 2001. A rapid and reli-able 7-deletion multiplex polymerase chain reaction assay foralpha-thalassemia. Blood 98: 250–251.

16. Lyke KE, Laurens M, Adams M, Billingsley PF, Richman A,Loyevsky M, Chakravarty S, Plowe CV, Sim BK, Edelman R,Hoffman SL, 2010. Plasmodium falciparum malaria challengeby the bite of aseptic Anopheles stephensi mosquitoes: resultsof a randomized infectivity trial. PLoS ONE 5: e13490.

17. Laurens MB, Billingsley P, Richman A, Eappen AG, AdamsM, Li T, Chakravarty S, Gunasekera A, Jacob CG, SimBK, Edelman R, Plowe CV, Hoffman SL, Lyke KE, 2013.Successful human infection with P. falciparum using threeaseptic Anopheles stephensi mosquitoes: a new model forcontrolled human malaria infection. PLoS ONE 8: e68969.

18. Epstein JE, Tewari K, Lyke KE, Sim BK, Billingsley PF, LaurensMB, Gunasekera A, Chakravarty S, James ER, Sedegah M,Richman A, Velmurugan S, Reyes S, Li M, Tucker K, AhumadaA, Ruben AJ, Li T, Stafford R, Eappen AG, Tamminga C,Bennett JW, Ockenhouse CF, Murphy JR, Komisar J, ThomasN, Loyevsky M, Birkett A, Plowe CV, Loucq C, Edelman R,Richie TL, Seder RA, Hoffman SL, 2011. Live attenuatedmalaria vaccine designed to protect through hepatic CD8+Tcell immunity. Science 334: 475–480.

19. Seder RA, Chang LJ, Enama ME, Zephir KL, Sarwar UN,Gordon IJ, Holman LA, James ER, Billingsley PF, GunasekeraA, Richman A, Chakravarty S, Manoj A, Velmurugan S, Li M,Ruben AJ, Li T, Eappen AG, Stafford RE, Plummer SH,Hendel CS, Novik L, Costner PJ, Mendoza FH, SaundersJG, Nason MC, Richardson JH, Murphy J, Davidson SA,Richie TL, Sedegah M, Sutamihardja A, Fahle GA, LykeKE, Laurens MB, Roederer M, Tewari K, Epstein JE, SimBK, Ledgerwood JE, Graham BS, Hoffman SL, for the VRC312 Study Team, 2013. Protection against malaria by intra-venous immunization with a nonreplicating sporozoite vaccine.Science 341: 1359–1365.

20. Adegnika AA, Verweij JJ, Agnandji ST, Chai SK, BreitlingLP, Ramharter M, Frolich M, Issifou S, Kremsner PG,Yazdanbakhsh M, 2006. Microscopic and sub-microscopic

Plasmodium falciparum infection, but not inflammation causedby infection, is associated with low birth weight. Am J TropMed Hyg 75: 798–803.

21. Nieman AE, de Mast Q, Roestenberg M, Wiersma J, Pop G,Stalenhoef A, Druilhe P, Sauerwein R, van der Ven A, 2009.Cardiac complication after experimental human malariainfection: a case report.Malar J 8: 277.

22. van Meer MP, Bastiaens GJ, Boulaksil M, de Mast Q, GunasekeraA, Hoffman SL, Pop G, van der Ven AJ, Sauerwein RW, 2014.Idiopathic acute myocarditis during treatment for controlledhuman malaria infection: a case report.Malar J 13: 38.

23. Anderson TJ, Su XZ, Bockarie M, Lagog M, Day KP,1999. Twelve microsatellite markers for characterizationof Plasmodium falciparum from finger-prick blood samples.Parasitology 119: 113–125.

24. Shaukat AM, Gilliams EA, Kenefic LJ, Laurens MB,Dzinjalamala FK, Nyirenda OM, Thesing PC, Jacob CG,Molyneux ME, Taylor TE, Plowe CV, Laufer MK, 2012.Clinical manifestations of new versus recrudescent malariainfections following anti-malarial drug treatment. Malar J11: 207.

25. Roestenberg M, de Vlas SJ, Nieman AE, Sauerwein RW,Hermsen CC, 2012. Efficacy of preerythrocytic and blood-stage malaria vaccines can be assessed in small sporozoitechallenge trials in human volunteers. J Infect Dis 206: 319–323.

26. Harteveld CL, Higgs DR, 2010. Alpha-thalassaemia. OrphanetJ Rare Dis 5: 13.

27. Taylor SM, Parobek CM, Fairhurst RM, 2012. Hemoglobinopa-thies and the clinical epidemiology of malaria: a systematicreview and meta-analysis. Lancet Infect Dis 12: 457–468.

28. Baliraine FN, Afrane YA, Amenya DA, Bonizzoni M, Vardo-Zalik AM, Menge DM, Githeko AK, Yan G, 2010. Acohort study of Plasmodium falciparum infection dynamicsin Western Kenya Highlands. BMC Infect Dis 10: 283.

29. Schultz L, Wapling J, Mueller I, Ntsuke PO, Senn N, Nale J,Kiniboro B, Buckee CO, Tavul L, Siba PM, Reeder JC, BarryAE, 2010. Multilocus haplotypes reveal variable levels ofdiversity and population structure of Plasmodium falciparumin Papua New Guinea, a region of intense perennial trans-mission. Malar J 9: 336.

30. Griffing SM, Mixson-Hayden T, Sridaran S, Alam MT,McCollum AM, Cabezas C, Marquino Quezada W, BarnwellJW, De Oliveira AM, Lucas C, Arrospide N, Escalante AA,Bacon DJ, Udhayakumar V, 2011. South American Plasmodiumfalciparum after the malaria eradication era: clonal popula-tion expansion and survival of the fittest hybrids. PLoS ONE6: e23486.

31. Mobegi VA, Loua KM, Ahouidi AD, Satoguina J, NwakanmaDC, Amambua-Ngwa A, Conway DJ, 2012. Population geneticstructure of Plasmodium falciparum across a region of diverseendemicity in West Africa.Malar J 11: 223.

32. Yalcindag E, Elguero E, Arnathau C, Durand P, Akiana J,Anderson TJ, Aubouy A, Balloux F, Besnard P, BogreauH, Carnevale P, D’Alessandro U, Fontenille D, GamboaD, Jombart T, Le Mire J, Leroy E, Maestre A, Mayxay M,Menard D, Musset L, Newton PN, Nkoghe D, Noya O,Ollomo B, Rogier C, Veron V, Wide A, Zakeri S, CarmeB, Legrand E, Chevillon C, Ayala FJ, Renaud F, PrugnolleF, 2012. Multiple independent introductions of Plasmodiumfalciparum in South America. Proc Natl Acad Sci USA 109:511–516.

480 SHEKALAGHE AND OTHERS

Supplemental Table 1

Results of potency and sporozoite membrane integrity assays (SMIA) on the lot of PfSPZ Challenge used in the clinical trial in Bagamoyo,Tanzania*

Time pointPotency

(no. of parasites expressing PfMSP-1/well)% Viability

(sporozoite membrane integrity assay)

Fresh 32.7 ± 1.5 98.2%Release 29.3 ± 3.1 87.4% ± 5.9%3 Month 27.3 ± 0.6 84.6% ± 1.9%6 Month 26.7 ± 1.5 83.6% ± 5.5%Post-last clinical dose–Oxford13 9 Month 26.3 ± 2.5 86.3% ± 6.5%12 Month 27.3 ± 0.6 86.2% ± 1.3%Post-last clinical dose–Tanzania (18 month) 24.0 ± 1.7 81.7% ± 2.6%

*Fresh PfSPZ used for the lot of PfSPZ Challenge used in this clinical trial produced 10% more PfMSP-1-expressing parasites in this assay than did PfSPZ that had been cryopreservedfor several days (Release). At 18 months, several weeks after inoculation of the last volunteers in Bagamoyo, the PfSPZ had a 27% reduction in potency by this assay as compared withfresh PfSPZ. There was an 11% reduction in the results of the sporozoite membrane integrity of cryopreserved PfSPZ at the time of Release, as compared with fresh PfSPZ. At 18 months,several weeks after inoculation of the last volunteers in Bagamoyo, the PfSPZ had a 17% reduction in the SMIA as compared with fresh PfSPZ.

Supplemental Table 2

Normal ranges at BRTC Laboratory and corresponding modified FDA Guidelines for toxicity grading*Test Normal Range Grade 1 Grade 2 Grade 3 Grade 4

Sodium (meq/l)–hypernatremia 136–146 N/A 146–147 148–150 > 150Sodium (meq/l)– hyponatremia 132–135 130–131 125–129 < 125Potassium (meq/l)– hyperkalemia 3.5–5 5.1–5.2 5.3–5.4 5.5–5.6 > 5.6Potassium (meq/l)– hypokalemia N/A 3.3–3.4 3.1–3.2 < 3.1Creatinine (mmol/L) 53–106 107–150 151–177 178–221 > 221 or requires dialysisGlucose (mmol/L)–hyperglycemia 3.89–6.0 6.01–6.94 6.95–11.1 > 11.1Glucose (mmol/L)– hypoglycemia 3.61–3.88 3.05–3.60 2.50–3.04 < 2.50AST (U/L) 5–34 1.1–2.5 + ULN 2.6–5 + ULN 5.1–10 + ULN >10 + ULNALT (U/L) 0–55 1.1–2.5 + ULN 2.6–5 + ULN 5.1–10 + ULN >10 + ULNBilirubin (mmol/L) 0–6.1 1.1–1.5 + ULN 1.6–2.0 + ULN 2.0–3.0 + ULN > 3.0 + ULNTotal white blood count + 1,000/mL 3.2–11.7 2.5–3.1 1.5–2.5 1.0–1.5 < 1.0Neutrophil count + 1,000/mL 2.0–6.9 1.5–1.999 1.0–1.499 0.500–0.999 < 0.500Lymphocyte count + 1,000/mL 0.87–3.19 0.750–0.869 0.500–0.749 0.250–0.499 < 0.250Eosinophil count + 1,000/ mL 0–0.7 0.701–1.5 1.501–5.0 > 5.0Hemoglobin (g/L) 12.9–18.3 12.5–13.5 10.5–12.4 8.5–10.4 < 8.5Platelets + 1,000/mL 146–345 125–145 100–124 25–99 < 25

*Guidance for Industry - Toxicity Grading Scale for Healthy Adult and Adolescent Volunteers Enrolled in Preventive Vaccine Clinical Trials – 2007 (http://www.fda.gov/downloads/BiologicsBloodVaccines/GuidanceComplianceRegulatoryInformation/Guidances/Vaccines/ucm091977.pdf). AST = aspartate aminotransferase; ALT = alanine aminotransferase; ULN =upper limit of normal.

Supplemental Table 3

Laboratory abnormalities that developed after administration of normal saline or PfSPZ Challenge from Day 5 through Day 28 post inoculation

Normal saline controls(N = 6)

1st day AE noted*(maximum Grade)†

10,000 PfSPZ(N = 12)

1st day AE noted(maximum Grade)*

25,000 PfSPZ(N = 12)

1st day AE noted(maximum Grade)*

Increased AST 6 (2), 14 (2) 9 (1), 9 (2), 12 (1), 12 (2) 5 (3), 14 (3), 15 (1), 18 (2)Increased ALT 14 (3), 15 (1) 9 (2), 12 (1) 5 (2), 14 (2), 15 (1)Increased Bilirubin 9 (2)Increased Creatinine 18 (1)Hypoglycemia 5 (2), 28 (1) 15 (1) and 19 (1), 15 (1), 15 (1) 5 (2) and 12 (3)Hyperglycemia 5 (2) 5 (2), 15 (1) 5 (1), 5 (2), 11 (1), 13 (2), 13 (2), 19 (2)Leukopenia 18 (1)Leukocytosis 14 (1)Neutropenia 5 (1) and 18 (1), 6 (1) and 28 (2),

9 (2), 27 (1)5 (2), 5 (2), 9 (1), 12 (1) and

18 (1), 12 (1), 27 (1), 28 (1)5 (2), 12 (1), 27 (2)

Lymphopenia 21 (1) 15 (1), 18 (2) 14 (1), 14 (3), 15 (1)Eosinophilia 5 (1)Anemia 28 (1), 28 (1) 11 (1), 15 (2)Thrombocytopenia 6 (1) 5 (2) and 12 (2), 9 (2), 13 (3), 18 (1) 15 (1)Proteinuria 5 (3), 5 (2), 12 (2), 12 (2), 28 (3) 5 (2), 9 (2)Hematuria 28 (2)

*Each entry represents a specific, different laboratory abnormality.†For those designated “and”, the abnormality occurred, resolved, and recurred.AE = adverse event; AST = aspartate aminotransferase; ALT = alanine aminotransferase.